Microwave Irradiation Induced Changes in Protein Molecular

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Microwave Irradiation Induced Changes in Protein Molecular Structures of Barley Grains: Relationship with Changes in Protein Chemical Profile, Protein Subfractions and Digestion in Dairy Cows Xiaogang Yan, Nazir Ahmad Khan, Fangyu Zhang, Ling Yang, and Peiqiang Yu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 18 Jun 2014 Downloaded from http://pubs.acs.org on June 29, 2014

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Journal of Agricultural and Food Chemistry

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Microwave Irradiation Induced Changes in Protein Molecular Structures of Barley

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Grains: Relationship with Changes in Protein Chemical Profile, Protein Subfractions

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and Digestion in Dairy Cows

4 Xiaogang Yan,*,† Nazir A. Khan,*,1 Fangyu Zhang,*,† Ling Yang,* Peiqiang Yu*,‡,2

5 6 7 8 9 10 11

*

12

Canada S7N 5A8

13

†

14

Gongzhuling, Jilin, China, 136100

15

‡

16

300384.

Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, SK,

The Branch Academy of Animal Science, Jilin Academy of Agricultural Science,

College of Animal Science and Animal Veterinary, Tianjin Agricultural University, Tianjin

17 18 19

1

Current address: Department Animal Nutrition, the University of Agriculture Peshawar,

20

Pakistan

21

2

22

and Bioresources, University of Saskatchewan, Saskatoon, SK, Canada S7N 5A8 Tel: +1 306

23

966 4132

24

Email: [email protected]

Corresponding author at: Department of Animal and Poultry Science, College of Agriculture

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ABSTRACT: The objectives of this study were to evaluate microwave irradiation (MIR)

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induced changes in crude protein (CP) subfraction profiles; ruminal CP degradation

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characteristics and intestinal digestibility of rumen undegraded protein (RUP); and protein

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molecular structures in barley (Hordeum vulgare) grains. Samples from hulled (n=1) and

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hulless cultivars (n=2) of barley, harvested from four replicate plots in two consecutive years,

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were evaluated. The samples were either kept as raw or irradiated in a microwave for 3 min

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(MIR3) or 5 min (MIR5). Compared to raw grains, MIR5 decreased the contents rapidly

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degradable CP subfraction (45.22 to 6.36% CP) and the ruminal degradation rate (8.16 to

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3.53% /h) of potentially degradable subfraction. As a consequence the effective ruminal

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degradability of CP decreased (55.70 to 34.08% CP), and RUP supply (43.31 to 65.92% CP)

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to the post-ruminal tract increased. The MIR decreased the spectral intensities of amide 1,

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amide II, α-helix and β-sheet, and increased their ratios. The changes in protein spectral

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intensities were strongly correlated with the changes in CP subfractions and digestive

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kinetics. Our results shows that MIR for a short period (5 min) with a lower energy input can

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improve the nutritive value and utilization of CP in barely grains.

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KEY WORDS: barley grain, microwave irradiation, heat processing, nutrient availability,

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protein molecular structure

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INTRODUCTION

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Barley (Hordeum vulgare) is the fourth largest cereal grain produced in the world, and

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predominantly (70%) used as animal feed.1 Canada is the sixth largest barley producer in the

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world, with an annual barley production of 7.76 million tonnes.2 In western Canada, barley is

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extensively grown for dairy cattle feeding, due to a short growing season and unfavorable

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(cooler) climatic conditions for corn (Zea mays L.) production.3 Barley is also the major

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ingredient (up to 90%) of the concentrates fed to beef cattle in western Canada. However, due

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to high fibre (hulled) content, the metabolizable energy content of hulled-barley is lower

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compared to corn, and can potentially impair the productivity of high producing animals.4

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Over the past few decades feed type hulless barley, characterized by the spontaneous loss of

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hulls during harvest, have been developed with an improved nutritive value such as lower

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content of fiber, and higher contents of crude protein (CP) and starch.5

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Barley is primarily used as energy source in dairy cow rations. Because of its high

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consumption and relatively high CP content, barley often strongly contributes to the CP

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intake of dairy cows. However, (hulless)-barley rapidly and extensively degrades in the

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rumen, resulting in a lower rumen undegraded protein (RUP) value.1,6 Traditionally, heat

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processing has been used to decrease the rapid degradation of plant-seeds derived CP in the

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rumen. However, the effects of heat processing on CP degradation in the rumen are

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equivocal,7,8 because heating conditions (temperature and duration) are often not optimal.

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Moreover, in the conventional heating systems, heat often penetrates from the surface to the

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interior of feed, as a result the outer side is often cooked (overheated) and the inside remains

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raw (under heated). Recently, microwave irradiation (MIR) based heat processing has

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received a remarkable acceptance in the feed industries, because it requires lower energy

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inputs and heat is uniformly distributed in the feed. Short period (3-8 min) MIR-induced

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heating has been shown to reduce soluble CP (SCP) and in situ CP degradation in canola

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meal,9 canola seeds,10 cottonseed meal,11 and corn grains.12 However, information on the MIR

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induced changes in intestinal digestibility of RUP is scarce. To the author’s knowledge, no

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systematic study has been conducted on the MIR induced changes in feed protein inherent

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molecular structures in relation to CP nutritive value and digestion in the rumen and intestine

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of dairy cattle.



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Heat processing reduces protein degradation in the rumen by altering protein

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molecular structures such as the uncoiling pleated structure and denaturation of the whole

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protein molecule.7 These structural changes exposes hydrophobic amino acids and also

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results in the development of cross-linkages between amino acids and reducing sugars or

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other amino acids/polypeptides, reducing their solubility and rumen degradation. Recent

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studies have shown that quantifying the heat-induced changes in protein inherent molecular

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structures is vital to understand the changes in their nutritive value, digestion and utilization

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in dairy cow,13-15 and establish optimal heating condition.7 Our recent studies have shown

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that Fourier transform/infrared-attenuated total reflectance (FT/IR-ATR) molecular

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spectroscopy can be used as a rapid, direct, non-destructive bioanalytical technique to detect

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heat-induced changes in protein molecular structures. 7, 15 The objectives of the present study

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were to reveal MIR induced changes in protein molecular structures of hulless and hulled

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barley, and correlated these structural changes to changes in 1) chemical profile; 2) CP

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subfractions composition and; 3) in situ rumen CP degradation and intestinal digestibility of

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RUP. We hypothesized that the MIR for different duration (3 or 5 min) will alter protein

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molecular structures to different extent in hulled and hulless barley, which will correspond to

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different level of alteration in protein nutritive value and digestibility.

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MATERIALS AND METHODS

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Samples Preparation and Treatment

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Grains from two newly-developed hulless cultivars (CDC-Fibar and HB08302) with altered

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starch composition (amylose/amylopectin ratio), and a normal hulled cultivar (Copeland) of

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barley were evaluated in present study. The samples were provided by Dr. Aaron D. Beattie

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(Crop Development Center, University of Saskatchewan, Saskatoon, Canada). The CDC-


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Fibar is a zero-amylose (0% amylose and 100% amylopectin) waxy cultivar, and HB08302 is

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a high-amylose (40% amylose and 60% amylopectin) cultivar. The Copeland is a normal 2-

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row malting barley cultivar. Grains were sampled (~2 kg) from four replicate plots of each

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cultivar grown at the Kernen Crop Research Fields (52°9' N and 106°32' W) of Crop

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Development Center (CDC; University of Saskatchewan, Saskatoon, SK, Canada) during two

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consecutive years (2009 and 2010). For each cultivar, eight samples were used for heat

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processing. A subsample (~500 g) of each sample were heated in microwave oven

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(DMW904W, Findlay, Ohio, US), operated at a power of 900 W (1.33 W/g) and irradiation

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frequency of 2450 MHz, for 3 (MIR3) or 5 (MIR5) min in two replicate runs. Raw grains

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(MIR0) were used as control.

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The control and heat treated samples were ground (Retsch ZM-1, Brinkmann Instruments

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Ltd., Mississauga, ON, Canada) through 0.25mm screen for molecular spectral analysis;

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through 2 mm for in situ rumen incubation studies and; through 1mm screen for all other wet

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chemical analysis.

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Chemical Analysis

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The ground (1 mm) samples of barley were analysed for the contents of DM (method

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930.15), ash (method 942.05), CP (method 984.13; using a KjeltecTM 2400 autoanalyzer;

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Foss Analytical A/S, Hillerød, Denmark) and ether extract (EE; method 920.39) according to

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the standard procedures of AOAC.16 The contents of neutral detergent insoluble CP (NDICP)

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and acid detergent insoluble CP (ADICP) were analyzed according to the method of Licitra et

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al.17 The content of SCP was analyzed according to the method of Roe et al.18 with slight

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modification as described by Peng et al.7 The non-protein nitrogen (NPN) content was

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analyzed according to the method of Licitra et al.17 The true protein in feeds were precipitated

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with tungstic acid and the NPN content was calculated as the difference between total CP



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content and CP content of the residues after filtration.

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Fractionation of Crude Protein

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The CP content of the feeds was fractionated into immediately solubilised protein (PA),

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potentially degradable true protein (PB) and unavailable true protein (PC) according to the

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Cornell Net Carbohydrate Protein System (CNCPS).19 The subfraction PB was further

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fractionated into three subfractions that are believed to have different rates of ruminal

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degradation. The PB1 is the rapidly degradable subfraction with a degradation rate of 120-

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400%/h; PB2 is the intermediately degradable subfraction with a degradation rate of 3-16%/h

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and; PB3 is the slowly degradable subfraction with a degradation rate of 0.06-0.55%/h.21 The

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PA is calculated as NPN × 6.25; subfraction PB1 is the buffer soluble protein and calculated

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as SCP – PA; subfraction PB2 is calculated as CP – (PA + PB1 + PB3 + PC); subfraction

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PB3 is calculated as NDICP – ACDIP and; subfraction PC is the ADICP.

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In Situ Rumen Incubation

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Three lactating Holstein dairy cows, each fitted with flexible rumen cannula with an internal

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diameter of 10 cm (Bar Diamond Inc., Parma, ID, USA), were used for the in situ rumen

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incubation studies. The fistulated cows were handled according to the guidelines of the

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Canadian Council on Animal Care.20 The cows were individually fed a balanced TMR (15

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kg/head/d on DM basis), containing 510 g barley silage, 150 g chopped alfalfa hay and 340 g

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concentrate per kg DM. The concentrate contained 560 g barley, 50g wheat, 50 g oats, 330 g

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dairy supplement pellets and 10 g molasses per kg DM. Whereas, the pellet dairy mixture

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contained 316 g soybean meal, 270 g canola meal, 100 g of peas, 85 g of premix (University

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of Saskatchewan), 55 g corn gluten meal, 55 g corn grain, 47 g sodium bicarbonate, 30 g

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ground barley, 20 g canola oil, 16 g of salt and 7 g Dynamate (K and Mg sulfate, University


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of Saskatchewan) per kg DM. The TMR was formulated according to NRC requirements for

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dairy cow,21 and fed twice daily (8:00 and 16:00) in equal portions. All cows had 24 h/d

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access to fresh drinking water.

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The in situ nylon bag technique as describe by Peng et al.,7 was used to determine the

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rumen degradation kinetics of the feeds. Briefly, 7 g of the coarsely ground (2 mm) sample of

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each treatment of each barley cultivar was weighed into pre weighed and coded nylon bags

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(10 × 20 cm) with a pore size of 41 µm (Nitex 03-41/31 monofilament open mesh fabric,

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Screentech Corp., Mississauga, ON, Canada). The ratio of sample-size to bag surface-area

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was 19 mg/cm2. The gradual addition, all out method was used. The bags were randomly

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incubated for 48, 24, 12, 8, 4, 2 and 0 h in rumen of the 3 fistulated cows in 2 replicate runs.

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To get enough residues for chemical analysis multiple bags were used. The multi-bags for 48,

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24, 12, 8, 4, 2 and 0 h incubation periods were 5, 4, 4, 2, 2, 2 and 2, respectively. A large

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polyester mesh bag (45 × 45 cm, fixed to rumen cannula with a 90 cm long rope) was used to

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hold the bags in rumen. To hold the bags in bottom liquid phase of the rumen a 250 mL

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plastic bottle filled with gravel was placed in the polyester mesh bag. The bags were removed

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from the rumen at the end of incubation period, and immediately gently rinsed (6 times) in a

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bucket

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microbial/enzymatic degradation. The 0 h samples were washed under similar conditions

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without rumen incubation. The bags were subsequently dried at 55ºC for 48 h, cooled, and

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reweighed. Residues were pooled together as per incubation time for each run. The dried

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residues were grounded and stored at 4ºC for chemical analysis.

with

cold

tap

water

to

remove

adhering

ruminal

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Rumen Degradation Kinetics


contents

and

stop


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The rumen degradation characteristics of DM and CP were computed according to the

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first-order kinetics equation of Ørskov and McDonald22 with slight modifications as proposed

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by Tamminga et al. 23 Y = U + 100 − S − U × e

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where Y (t) is the DM/CP (%) present in the residues at t hours of incubation; S is the soluble

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subfraction (%); U is the rumen undegradable subfraction (%); Kd is the degradation rate of

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potentially degradable subfraction (%/ h) and; T0 is lag time (h). The in situ parameters were

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analyzed using the nonlinear (PROC NLIN) procedure with iterative least-squares regression

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(Gauss-Newton method) of the Statictical Analysis System (SAS).24 The effective

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degradability of DM (EDDM) and CP (EDCP) was computed according to the NRC-2001

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dairy model21 as follow, EDDM/CP = S + D ×

K

K + K

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where D is the potentially degradable subfraction (%) and estimated as: D = 100 – S – U, and

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Kp is the estimated outflow rate of digesta from rumen, which was assumed to be 6%/h.23 The

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rumen undegraded DM and CP was calculated as follow RUDM/CP = U + D ×

K

K + K

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Intestinal Digestibility of Rumen Undegraded Protein

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The 3-steps in vitro procedure as described by Calsamiglia and Stern25 was used to

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determine the intestinal digestibility of RUP. Briefly, a calculated amount of dried ground

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residues of 16 h rumen incubation, containing approximately 15 mg of N, were transferred to

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15-mL centrifuge tubes. Subsequently, 10 mL of 0.1 N HCL solution containing 1 g/L pepsin

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was added to the residues. The mixtures were then vortexed, and incubated in shaking water

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bath at 38ºC for 1 h. After incubation, 0.5 mL of 1 N NaOH was added to mixture to 8 ACS Paragon Plus Environment

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neutralize the pH. Subsequently, 13.5 mL of phosphate buffer (pH 7.8) containing 37.5 mg of

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pancreatin was added to the mixture, vortexed, and further incubated at 38ºC for 24 h. After

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incubation, 3 mL of 100% (wt/vol) TCA solution was immediately added to precipitate the

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undigested protein and stop enzymatic reactions. The mixtures were vortexed, and then

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centrifuged at 10,000 × g for 15 minutes at a room temperature (20-22ºC). The supernatant

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was collected and analyzed for (soluble/digested) N content. Duplicate blank (without

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sample) tubes were run to correct for background effect of the solutions and enzymes

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mixtures used during the analysis. The intestinal digestibility of RUP was computed by

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dividing the content of TCA-soluble N by the total N in the residue sample.

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Molecular Spectroscopy

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The protein molecular spectral analyses were carried out at the molecular

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spectroscopy laboratory of the department of Animal and Poultry Science, University of

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Saskatchewan (Saskatoon, Canada). The molecular spectral profiles were generated using a

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JASCO FT/IR-ATR-4200 (Jasco Inc., Easton , MD), equipped with a deuterated L-alanine

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doped triglycine sulfate detector and a ceramic IR light source, and fitted with MIRacleTM

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ATR accessory module with a ZnSe crystal and pressure clamp (Pike Technologies, Madison,

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WI, USA). The spectra were generated in a transmission mode in the mid-IR (ca. 4,000-800

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cm-1) and amide fingerprint (ca. 1732-1483 cm-1) regions, with 128 co-added scans and a

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spectral resolution of 4 cm-1. Each sample was run 5 times. The spectra were collected,

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corrected for background and CO2 noise using Jasco Spectra Manager II software. The

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protein primary and secondary structures related molecular spectral features from the FT/IR

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spectra were quantified using Ominic 7.3 software (Spectra-Tech Inc., Madison, WI, USA).

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The spectral bands of the protein functional groups (C=O, C−N, and N−H), representing the

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primary molecular structures, were identified and assigned using published information.7, 26



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Briefly, the amide-I (∼80% C=O and ∼10% C−N stretching vibration, and 10% N−H

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bending vibration; centered at a wavelength of ca. 1,655 cm-1) and amide-II (∼60% N−H

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bending vibration, ∼40% C−N stretching vibration; centered at ca. 1,565 cm−1) were

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quantified for the characterization of protein primary structures. For characterization of

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protein secondary structures, FSD spectrum was generated using Fourier self-deconvolution

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(FSD) or the secondary derivative functions in the OMNIC software. The FSD spectrum was

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further resolved into several multicomponent peaks in the protein amide I region, where α-

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helices (centred at ca. 1653 cm-1) and β-sheets (centred at ca. 1630 cm-1) were identified.

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Peak area, height of the spectral intensities of amide-I and amide-II, and α-helix and β-sheet

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were measured and ratios were calculated.

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Statistical Analysis

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Data on chemical profiles, CP subfractions, digestible nutrients, energy values FT/IR

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spectra, in situ rumen degradation characteristics and intestinal digestibility of RUP were

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analyzed according to the PROC MIXED procedure of Statistical Analysis System (SAS).24

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The model used for analysis was: #$%& = ' + ()*$ + +,% + -$%&

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where Yijkl is an observation on the dependent variable ijkl; µ is the overall population mean;

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MIRi, is the fixed effect of MIR (control, 3 and 5 min irradiation); BCj, is the fixed effect of

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the barley cultivars (CDC Fibar, HB08302 and Copeland); and eijk is random error associated

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with the observation ijk. Except for few parameters, the interactions of MIR and barley

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variety were non-significant and hence removed from the final model. Significant (P < 0.05)

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interactions are reported in the footnote of Tables. Post-hoc analyses were carried out using

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the Tukey-Kramer test to compute pair-wise differences in the means. Means with different

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superscript letter groups were obtained with “pdmix 800 SAS macro”.27 The PROC CCORR


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of SAS24 was used to determine the strength of relationship between protein molecular

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structural characteristics and the chemical profiles, CP subfractions, in situ rumen

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degradation characteristics and intestinal digestibility of RUP.

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The agglomerative hierarchical cluster analysis (CLA) and principal component analysis

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(PCA) were performed on the spectral data at the fingerprint region ca. 1732-1483 cm-1

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(representing amides I and II bands) to visualize and classify the difference in intrinsic

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protein molecular structures of the feeds using Statistica software (Version 8, StatSoft Inc.,

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Tulsa, OK). The detailed concepts and procedure of using CLA and PCA for FTIR/ATR

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spectral analysis is described earlier.13

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RESULTS AND DISCUSSION

254 255

Microwave Irradiation Induced Changes in Chemical Profile

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Results on the MIR induced changes in the mean chemical profile of different cultivars of

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barley are shown in Table 1. Compared with the raw grains, MIR increased (P < 0.001) the

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content of DM, indicating that MIR decreased the moisture holding capacity of barley grains.

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Previous studies have shown a decrease in DM content with dry heating of Camelina7 and

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moist heating of flaxseed.14 Compared with the raw grains, the contents of NDICP and

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ADICP increased with the increasing duration of MIR. Samadi and Yu15 and Penge et al.7

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also observed an increase in cellwalls bound CP with moist heating (60 min at 120ºC) of

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soybean and camelina seeds, respectively. The increase in fiber bounded CP could be related

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to the heat induced cross-linkages between amino acids and reducing sugars such as during

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Maillard reactions.7 This is further supported by the consistent increase in The MIR markedly

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decreased (50.84 to 17.56% CP) the SCP content of barley. This is in agreement with

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previous findings of a marked decrease in the SCP subfractions of camelina seeds (52.73 to



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20.41% CP)7, soybeans (43.38 to 11.35% CP)15 and flaxseeds (51.88 to 8.82% CP)14 with

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moist heating (at 120ºC for 60 min). In contrast, the NPN content increased (P < 0.001) with

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MIR. The present result is supported by the findings of Doiron et al.14 and Peng et al.7

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However, Mustafa et al.28 and Samadi and Yu15 observed a decrease in NPN content during

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heat processing. This discrepancy may be related to the differences in heating methods (MIR

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vs. dry toasting, moist heating) or to the inherent differences in seed types (cereal grains vs.

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oilseeds) and NPN methodologies (tungstic acid vs. TCA, differences in filter papers pore

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size etc.). Overall, our present findings suggest that a short term (5 min) MIR induces more

276

or less similar changes in the CP profiles of feed as have been reported for other feeds after

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60 min dry/moist heating at 120°C. Except SCP for which the interaction was significant

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(results not shown), the MIR induced changes in chemical profile were independent of barley

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types. As discussed in detailed earlier5 the hulless barley cultivars had a higher (P < 0.001)

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contents of CP and EE than the hulled barley.

281 282

Microwave Irradiation Induced Changes in Crude Protein subfractions

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Results on the MIR-induced changes in CP subfractions are presented in Table 2. The

284

contents of all CP subfractions in barley grains altered (P < 0.05) with MIR, and PB1 was the

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only subfraction that decreased (P < 0.05) in concentration. The MIR for 3 min increased (P

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< 0.05) the PA (soluble) subfraction, with further irradiation for 5 min had no significant

287

effect on PA subfraction. The major changes in CP subfractions, however, occurred in the

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contents of PB1 and PB2. The PB1 (rapidly degradable) subfraction markedly decreased

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from 45.22 to 6.36% (%CP), whereas the PB2 (intermediately degradable) subfraction

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markedly increased from 35.84 to 59.43% (%CP). The increase (5.62 to 11.19% of CP) in PA

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(NPN × 6.25) subfraction with MIR was quantitatively small. Therefore, the shift from

292

ruminally degradable PB1 subfraction to a partially ruminally degradable PB2 subfraction


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demonstrates an overall decrease in ruminal CP degradation rate in the irradiated barley

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grains. Although the changes in CP-subfractions with MIR has not been evaluated previously.

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The changes in PB1 and PB2 subfractions after MIR (for 5 min) were comparable with the

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changes produced by moist heating at 120ºC for 60 min.7, 14 The moist heating decreased the

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PB1 subfraction of flaxseed from 45.9 to 10.5% CP with a concomitant increase in the PB2

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subfraction from 43.0 to 73.3% CP.14 Similarly the moist heating of camelina seeds decreased

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the PB1 subfraction from 45.06 to 16.69% CP and increased PB2 subfraction from 45.28 to

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70.58% CP.29 Compared to raw grains MIR for 3 min did not change PB3 and PC

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subfractions. However, MIR for 5 min significantly increased (P < 0.05) the PB3 and PC

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subfractions. The PB3 is believed to be more slowly degraded in the rumen, and large portion

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of this subfraction escapes ruminal degradation and digested in the small intestine. Therefore,

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the increase in PB3 subfraction will contribute to the digestible RUP supply to the small

305

intestine. Subfraction PC is considered non degradable in the rumen, and CP in this

306

subfraction is heat damaged or bound to other compounds such as lignin, tannins and

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Maillard reaction products.19 The MIR for 3 min decreased (P < 0.05) PB1 subfraction and

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increased (P < 0.05) PB2 subfraction but did not alter PC subfraction (Table 2). These results

309

suggest that MIR for 3 min decreased CP degradability in the rumen but did not overprotect

310

the CP from digestion in the small intestine. However, MIR for 5 minutes significantly

311

increased (0.97 to 5.32% CP) the PC subfraction, suggesting an overheating and protection of

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protein from intestinal digestion. These results show that MIR can decrease ruminal

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degradation of feed CP in relatively shorter time and much less energy inputs compared to

314

conventional heating systems.

315

The MIR induced changes in CP subfractions were independent of barley types.

316

Except PB1, which was lower (P < 0.001) in hulless barley, the mean contents of all other CP

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subfraction did not significantly differ due to barley type (hulless vs. hulled). Across the



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barley varieties, CDC-fibar had a higher (P < 0.05) PB2 subfraction and a lower (P < 0.05)

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PC subfraction. These findings are in general agreement with Yang et al.,5 who observed no

320

significant differences in the S and D subfractions of hulless and hulled barley. Whereas the

321

degradation rate was higher for the hulled (Copeland) cultivar.

322 323

Microwave Irradiation Induced Changes in Rumen degradation and Intestinal

324

Digestibility

325

Results on MIR induced changes in ruminal DM and CP degradation kinetics and intestinal

326

digestibility of CP are presented in Table 3. The MIR5 decreased (P < 0.05) the EDDM and

327

increased (P < 0.05) rumen undegraded DM. The S-subfraction of DM did not significantly

328

change and the D-subfraction increased (P < 0.01) with MIR5. Therefore, the decrease in

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EDDM of the irradiated barely could mainly be related to the marked decrease (13.08 to

330

7.37%/h) in the Kd of D-subfraction. In agreement with our findings, a 6 min MIR markedly

331

decreased the Kd of canola seeds (10.2 to 5.5%/h)10 cottonseed meal (7.6 to 4.7%/h)11, corn

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grain (5.1 to 4.3%/h)12 and canola meal (7.0 to 4.9%/h).9 The MIR3 did not significantly alter

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any of the DM and CP degradation parameter. In contrast there was a significantly shift from

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PB1 to PB2 CP subfraction after MIR3. This discrepancy may be, partly, explained by the

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higher kd of barley grain in the rumen. Although the S-subfraction of DM did not change

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with MIR5, the S-subfraction of CP consistently decreased (6.45 to 0.00%; P < 0.05) with the

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MIR5. This means that the S-subfraction of other nutrients than CP increased with MIR5.

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Starch represents the bulk of non-CP DM of barley grains, and previous studies have shown

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that MIR for 5 min increased the S-subfraction of starch in cereal grains,12,30 partly due to

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gelatinization of starch with heating.6 The decrease in S-subfraction of CP with 6 min MIR of

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corn grains,12 canola seeds,10 and cottonseed meal11 have been reported. The MIR5 decreased

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(P < 0.001) the EDCP with a concomitant increase (P < 0.001) in the supply of RUP to the


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Page 15 of 39


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post-ruminal tract (Table 3). The lower ruminal EDCP of the irradiated barley could be

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related to both lower S-subfraction and lower Kd of D-subfraction. Moreover, the changes in

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CP subfractions such as the decrease in rapidly degradable (PB1) subfraction and increase in

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intermediately degradable (PB2) and slowly degradable (PB3) subfractions could explain the

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lower ruminal EDCP.7, 15 These results demonstrate that a short term (5 min) MIR induced a

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major shift in the site of CP digestion from rumen to post-ruminal tract. Digestion of feed

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protein in the small intestine results in lower losses of protein and essential amino acids.

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Moreover, the gradual degradation of D-subfraction in the irradiated barley can optimize the

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efficiency of rumen microbial protein synthesis with a consequent decrease in NH3-N losses

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to the environment. However, overheating can reduce protein digestion in the post-ruminal

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tract. 15 In the present study, the MIR for 5 min significantly decrease (79.35 to 67.86%; P

Microwave Irradiation Induced Changes in Protein Molecular

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